王成花，孙诗晴，徐巨龙，赵小龙，薛超彬
山东农业大学植物保护学院/农药毒理与应用技术省级重点实验室，山东泰安 271018
Differential Expressed Genes and their Pathways of the Resistance to Flubendiamide in Plutella xylostella
WANG ChengHua, SUN ShiQing, XU JuLong, ZHAO XiaoLong, XUE ChaoBin
College of Plant Protection, Shandong Agricultural University/Provincial Key Laboratory of Pesticide Toxicology and Application Technique, Taian 271018, Shandong
摘要 【目的】随着氟苯虫酰胺等双酰胺类杀虫剂的广泛应用，小菜蛾（Plutella xylostella）对该类药剂的抗性愈发突出。研究旨在从转录组水平研究小菜蛾对氟苯虫酰胺的抗性机制，明确小菜蛾对氟苯虫酰胺产生抗性的关键基因及通路，为揭示小菜蛾对氟苯虫酰胺的抗性机制打下基础。【方法】以室内筛选的小菜蛾抗氟苯虫酰胺品系（Rh28）、田间抗性种群（Rz36）和敏感品系（S）为材料，通过转录组测序技术（RNA-Seq），获得抗性小菜蛾品系/种群在转录水平上的差异表达基因及抗性显著上调表达基因，采用基因本体（GO）显著性富集分析及京都基因与基因组百科全书数据库（KEGG）富集分析，确定差异表达基因具有的主要生物学功能及其参与的主要生化代谢和信号转导途径。【结果】通过将不同抗性品系/种群与敏感品系的RNA-Seq数据对比，获得数量不等的差异表达基因。将差异基因富集的生物学过程（biological process）和信号通路（pathway）进行GO分析，发现其主要集中于对刺激物的反应（response to stimulus）、催化活性（catalytic activity）和代谢过程（metabolic process）等条目中；而KEGG分析发现，差异基因主要集中于代谢通路（metabolic pathway）中，基因数量占比最高，为17.84%。通过统计分析进一步获得了抗性显著上调表达基因，并对其中218个基因开展了GO分析，其仍然主要集中在代谢过程、对刺激物的反应和生物调节（biological regulation）等条目中；通过对抗性显著上调基因进行KEGG分析，发现其仍主要集中在代谢通路中，其中谷胱甘肽-S-转移酶、细胞色素P450解毒酶及参与昆虫多种生理反应的热激蛋白（Hsp）超基因家族等基因的表达量均显著上调。通过聚类热图分析发现，显著表达上调的基因较多集中在内质网上蛋白质的加工（protein processing in endoplasmic reticulum）、酪氨酸代谢（tyrosine metabolism）、咖啡因代谢（caffeine metabolism）和Wnt信号通路（Wnt signaling pathway）中。【结论】获得了小菜蛾抗氟苯虫酰胺差异表达基因及抗性显著上调表达基因，其主要富集于代谢过程、应激反应及对刺激物的反应等条目中，这些基因的相互协同调控是小菜蛾对氟苯虫酰胺产生抗性的重要机制。
关键词 ： 转录组测序, 差异表达基因, 氟苯虫酰胺, 抗性, 小菜蛾
Abstract：【Objective】With the widespread use of diamide insecticides, such as flubendiamide, the resistance of Plutella xylostella to these insecticides became more and more prominent. The objective of this study is to research the resistant mechanism to flubendiamide in P. xylostella from the transcriptome perspective, identify the key genes and pathways that lead to resistance to flubendiamide in P. xylostella, and to lay a foundation for revealing the mechanism of resistance to flubendiamide in P. xylostella.【Method】 The resistance to flubendiamide in P. xylostella was determined by the RNA sequencing technique (RNA-Seq) using the flubendiamide-resistant strains (Rh28), the field resistant population (Rz36) and the susceptible strain (S). The differential expressed genes (DEGs) and significantly up-regulated resistant genes were obtained in the strains/populations. The enrichment analysis of Gene Ontology (GO) and the enrichment analysis of Kyoto Encyclopedia of Gene and Genome (KEGG) were used to identify DEGs. The major biological functions, the major biochemical metabolic and signaling pathways of the DEGs were elaborated. 【Result】The different numbers of DEGs were obtained by comparing RNA-Seq data of the different resistant strains/populations with susceptible strains. GO analysis of the biological process (BP) and pathway for differential gene enrichment showed that they were mainly focused on response to stimulus, catalytic activity and metabolic process items. KEGG analysis found that the differential genes were mainly concentrated in the metabolic pathway, with the highest number of genes (17.84%). the genes with significantly up-regulated expression were further obtained, and GO analysis was carried out on 218 of them, which still mainly focused on the metabolic process, response to stimulus and biological regulation items. KEGG analysis of significantly up-regulated expression genes found that the DEGs were still mainly in the metabolic pathway. The genes of glutathione-S-transferase, cytochrome P450 detoxification enzymes, and involved in a variety of insect physiological responses to heat shock protein (Hsp) family genes were significantly up-regulated compared with the susceptible strain. The results also showed that most of the genes with significant up-regulation were concentrated in protein processing in endoplasmic reticulum, tyrosine metabolism, caffeine metabolism and the Wnt signaling pathway by heat map and clustering analysis.【Conclusion】the DEGs and the significantly up-regulated expression genes were obtained in resistant strain of P. xylostella, which of them were mainly involved in the metabolic process, the response to stress and the response to stimulus. The synergistic regulation of these genes is an important mechanism of resistance to flubendiamide in P. xylostella.
Key words： RNA sequencing differential expressed genes flubendiamide resistance Plutella xylostella
收稿日期: 2017-12-27
基金资助:国家自然科学基金（31672046）、“双一流”奖补资金（SYL2017YSTD06）
通讯作者: 者薛超彬，E-mail：cbxue@sdau.edu.cn
作者简介: 王成花，E-mail：wangch0602@163.com
引用本文:
王成花，孙诗晴，徐巨龙，赵小龙，薛超彬. 抗氟苯虫酰胺小菜蛾差异表达基因及其通路[J]. 中国农业科学, 2018, 51(11): 2106-2115.
WANG ChengHua, SUN ShiQing, XU JuLong, ZHAO XiaoLong, XUE ChaoBin. Differential Expressed Genes and their Pathways of the Resistance to Flubendiamide in Plutella xylostella. Scientia Agricultura Sinica, 2018, 51(11): 2106-2115.
